Fluorescence detector, filter device and related methods

ABSTRACT

Microfluidic assay detectors and microfluidic assay detection methods are disclosed. A microfluidic chip is coupled to a light emitting device, emission filters and excitation filters. Excited fluorescent light is detected by a camera and a lens. The correspondent reading allows parallel detection of features such as antigens and biomarkers. A microfluidic filter and related methods are also disclosed. The filter can be used with on-chip fluid filtration such as whole blood filtration for microfluidic blood analysis. The filter is able to filter the necessary volume of fluid and in particular blood in an acceptable time frame.

RELATED APPLICATIONS

The present application claims the benefit of provisional application60/802,068 for “Personalized Medicine Device” filed on May 19, 2006 andincorporated herein by reference in its entirety. The presentapplication also claims the benefit of provisional application60/900,246 for “Microfluidic Device to Extract Blood Plasma from aFinger Stick” filed on Feb. 9, 2007, also incorporated herein byreference in its entirety. The present application is also related toU.S. patent application Ser. No. 11/439,288 “High ThroughputMulti-Antigen Microfluidic Fluorescence Immunoassays” filed on May 22,2006 and to U.S. patent application Ser. No. 11/297,651, “PrototypingMethods and Devices for Microfluidic Components”, filed on Dec. 7, 2005all of which are also incorporated herein by reference in theirentirety.

GOVERNMENT INTEREST

The U.S. Government has certain rights in this invention pursuant toDARPA grant no. HR0011-04-1-0032, NIH grant no. HG022644, and NIH, grantno. HG002644.

BACKGROUND

1. Field

The present disclosure relates to the field of sample analysis, inparticular to sample analysis performed for medical purpose. More inparticular, the present disclosure relates to a sample filter suitablefor separating sample components to be analyzed and to a fluorescencedetector suitable for detecting fluorescence signal resulting from afluorescence assay and related methods.

2. Related Art

In a fluorescence assay, the intensity of fluorescence is used to readthe concentration of the antigen, and is typically measured with afluorescence microscope or a laser scanner. The measured fluorescentintensity is typically compared to a previously measured “standardcurve” made from previous measurements of standard samples in order toascertain the concentration of antigen in the current sample. Thisserial measurement process is both time-consuming and expensive, andlimits the speed with which a fluorescence assay can be completed.

Many approaches for fluorescence assay test chips have been attempted sofar. Such approaches use glass, TiO₂, silicon, and silicone fluidics andhave so far demonstrated the opportunities of more complex fluidicsystems. In particular microfluidics technology provides the foundationfor advances in this field. Soft-Lithography allows for the cheap andefficient creation of polymer chips able to perform SandwichEnzyme-Linked ImmunoSorbent Assay (ELISA) tests on a microscopic scale.Such systems can run with a reduced amount of sample fluid, (e.g. lessthan a drop of patient blood serum) and synthesized proteins unlike thecurrent macroscopic versions which require significant amounts of both.

By reducing the size of devices, cost of manufacturing, and amount ofmaterial needed these chips prove essential in the creation of point ofcare medical testing. However, until now, the read-out mechanism forsuch chips has involved the use of rather large fluorescence microscopesor laser scanners.

The use of large size and high cost equipment impacts in particularperformance of fluorescence assays for medical purposes, in particulardiagnostic assays. The high cost and time requirements for medicaltesting restrict prompt detection and efficient treatment of ailments.Modern medical technology remains large and expensive, requiringcentralized healthcare systems which increase delays, price, and theprobability of clerical error and often cause prolonged hospital stays.

Ideally, more compact handheld devices are desirable in particular forpoint of care diagnostic assays that can evaluate fluorescence frommultiple chambers in parallel by using multi-element imaging detectingdevices.

Microfluidic testing also requires the extraction and analysis of smallquantities of patient's fluids. In particular, when the fluid is blood,working with on-chip microsystems for whole blood analysis, requiresprocessing the whole blood into components that can be analyzed usingmicrofluidic technology.

It is well known that cell inclusion may lead to cell lysis affectingthe reproducibility and standardization of blood tests. It is also wellknown that removing blood cells in an initial step can be importantsince miniaturized downstream systems, such as on-chip detectionmodules, protein analysis, PCR etc are prone to be clogged by cells andcoagulation. Blood filtration is necessary for all assays requiringplasma as well. Viral screening and other blood-type analysis may notdeal specifically with blood cells, but what else may be found inblood—such as proteins or antibodies. In that context, it is necessaryto filter whole blood.

In the context of microfluidic blood analysis, the system of bloodfiltration and anti-coagulation is necessary to handle blood samples ina miniaturized format. That is to say, there must be a way to separateblood cells and plasma from whole blood, on a microfluidic scale, forproper microfluidic blood analysis. Known filters in planarpoly(dimethylsiloxane) (PDMS) require registration and sealing betweentwo layers and the filter. This is difficult because only the thinnestof filters can be sealed this way, and leakage is a problem.

With the increased use of microfluidic technology in the fields ofphysics, chemistry, engineering, biotechnology, and especially medicine,it has become increasingly more important to discover more viable andmore efficient system of blood filtration and anti-coagulation, aprocedural practice for microscale whole blood analysis.

SUMMARY

According to a first aspect, an fluorescence assay detector is provided,comprising: an excitation filter; an emission filter; a microfluidicfluorescence assay apparatus located between the excitation filter andthe emission filter, the microfluidic fluorescence assay apparatuscomprising a fluorescence source; a light emitting device to excite thefluorescent source; a detecting arrangement for detecting fluorescencesignals or images on the microfluidic fluorescence assay apparatus.

According to a second aspect, a method for detecting fluorescence assaysignals is provided, comprising: providing excitation light at anexcitation frequency to excite a fluorophore, allowing the fluorophoreto emit light in connection with a microfluidic assay apparatus;filtering the excitation light; filtering light emitted through thefluorophore; and detecting fluorescence signals or images thusgenerated.

A first advantage of the detector and detecting method disclosed herein,is the possibility to perform fluorescence detection from microfluidicassay chips with digital imaging equipment reduced in size and able toobtain multiple assay readings in parallel.

A second advantage of detector and detecting method disclosed herein isthe possibility to provide equipment to perform fluorescenceidentification of many proteins in many blood samples, the equipmentreduced in size so that it is portable.

A third advantage of the detectors and detecting methods disclosedherein is that as a consequence of the reduction in size of theequipment a diagnostic tool resulting in improved cost,manufacturability and ease of operation can be constructed. The detectorand detecting method could be particularly useful in multi-analytehigh-throughput assay in several fields, including medical immunoassaytests.

According to a third aspect, a microfluidic filter forming mold isprovided, comprising: a top pin; a bottom pin connected with the top pinbut separable from the top pin, the bottom pin including an internalpin; a top piece with which the top pin is connected; and a bottom piecewith which the bottom pin is connected. The mold can further comprisethe microfluidic filter, which in a preferred embodiment is a bloodfilter.

According to a fourth aspect, a three-dimensional polymeric structurecomprising a polymeric cast of the above mold is provided. Thethree-dimensional polymeric structure can include a microfluidic filter.

The three-dimensional polymeric structure and of the single piecepolymeric structure of microfluidic filter device, can also beintegrated with microfluidic channeling structures associated to aninputting end and/or an outputting end of the structure. Themicrofluidic channeling structure on the outputting end is adapted tointerface with a microfluidic assay apparatus. The fluid would pass fromthe inputting end to the outputting end of the structure through thefilter embedded in the three-dimensional polymeric structure, separatingthe fluid component which would be used in the microfluidic assayapparatus.

According to a fifth aspect a microfluidic filtering device isdisclosed, the device comprising a microfluidic filter cast in a singlepiece polymeric structure, the microfluidic filter connected to aninputting end of the structure through a microfluidic inputting channel,the microfluidic filter connected to an outputting end of the polymericpiece structure through an outputting microfluidic channel.

The microfluidic filtering device, can also be integrated withmicrofluidic channeling structures associated to the inputting endand/or the outputting end of the structure according to what describedfor the three dimensional polymeric structure, mutatis mutandis.

According to a sixth aspect, a process for fabricating athree-dimensional fluid filtering device, in particular a bloodfiltering device, structure is provided, the process comprising:providing a mold having a top pin, a bottom pin and a blood filterbetween the top pin and the bottom pin; pouring polymer on the mold;curing the polymer; separating the top pin from the bottom pin to freethe blood filter; and extracting the mold from the polymer while leavingthe blood filter in the polymer.

An advantage of the filtering structures and devices in accordance withthe present disclosure over traditional planar microfluidics forfiltration in that a leak tight seal can be formed by virtue of the factthat the entire device is in one piece of polymer.

According to a seventh aspect a microfluidic filtering and detectingassembly, is disclosed, the assembly comprising: the three-dimensionalstructure or the microfluidic filtering device herein described; and thefluorescence assay detector of claim herein described.

According to a eight aspect a portable diagnostic system/assembly isdisclosed, the system comprising: a sample preparation stage: and asample analysis stage to detect analytes.

In the portable diagnostic system herein disclosed the samplepreparation stage can comprise a microfluidic circuit with a fluorescentfluid source, and the sample analysis stage can comprise: an excitationfilter; an emission filter; a light emitting device to excite thefluorescent fluid source; and a detecting arrangement for detectingfluorescence images from the microfluidic circuit, with the microfluidiccircuit located between the excitation filter and the emission filter.The detecting arrangement can also include means for comparing thefluorescence images detected to predetermined standard images to providea qualitative or a quantitative diagnostic indication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the device according to thepresent disclosure.

FIG. 2 shows a more detailed representation of the arrangement of FIG.1.

FIG. 3 shows a graph illustrating The emission and excitation curves ofAlexafluor 555 spinner from Laurel Technologies Corp.

FIG. 4A shows a bottom view of a microfluidic chip

FIG. 4B shows a fluorescent image of a microchannel.

FIGS. 5-7 show graphs of concentration of Alexa Fluor with respect tonormalized S/N.

FIG. 8 shows cancer markers and their clinically relevant concentrationswithin human blood.

FIG. 9A shows a perspective view of a mold used to form the microfluidicblood filter in accordance with the present disclosure.

FIG. 9B shows a cast microfluidic polymer structure with an embeddedfilter element obtained through use of the mold of FIG. 9A in a castingprocess, together with the mold of FIG. 10.

FIG. 10 shows a schematic view of an asymmetric porous membrane to beused as a blood filter for the present disclosure.

FIG. 11 shows a flow chart of the process to reach the structure of FIG.11 starting from the mold of FIG. 10.

FIG. 12 shows a perspective view of the PDMS blood filter of the presentdisclosure integrated with a microfluidic blood analysis chip and ablood draw capillary tube.

FIG. 13 shows a simulated side view of the blood filter device duringoperation.

FIG. 14 shows two microfluidic channels, one with anti-coagulated mouseblood flowing through it and the other with the filter directlyconnected to its input port.

FIG. 15A shows an unfiltered image of a microfluidic chip taken with aminiaturized detector of the disclosure.

FIG. 15B shows a schematic diagram of a microfluidic chip suitable inthe detector of the disclosure, wherein the control layer is shown indark gray, and the flow layer is shown in light grey.

FIG. 16A shows an image illustrative of the signal from CRMP5 positivechambers viewed from a microscope

FIG. 16B shows a saturated fluorescent signal as observed with aminiaturized detector of the disclosure

FIG. 17 shows graphs illustrating multiple sclerosis symptoms motifs.

FIG. 18 shows a schematic diagram of a microfluidic chip suitable in thedetector of the disclosure, wherein the control layer is shown in darkgray, and the flow layer is shown in light grey.

FIG. 19 shows the results of an MMP-9 Half Stack Immunoassay, whereinthe left panel shows the signal from the immunoassay and the right panelshows the control experiment.

FIG. 20 shows a selective three-layer protein stack for MMP-9.

FIG. 21 shows a fluorescence image confirming presence of MMP-9.

FIG. 22 shows a top view of a microfluidic chip suitable in the detectorof the disclosure.

DETAILED DESCRIPTION

According to a first embodiment a microfluidic fluorescence assaydetector and related methods are disclosed, the detectors to be used incombination with a microfluidic fluorescence assay apparatus, circuit orchip. In certain embodiments, the microfluidic fluorescence chip is ahigh-throughput multi-antigen fluorescence microfluidic assay chip, suchas immunoassay-chips made from PDMS or or other polymers such as SIFEL,which can provide quantitative blood analysis at clinically relevantlevels, such as 10-100 pM.

In Applicants' most recently developed immunoassay chips, activemicrofluidic matrix formats utilize arrays of integrated micromechanicalvalves to direct pressure-driven flow and to multiplex analyte sampleswith immunoassay reagents. ELISA-like fluorescence immunostacks areformed in the microchambers at the intersections of sample and reagentchannels. In the present disclosure, Applicants show that thefluorescence signals of the captured antigens from these microchamberscan be measured with a digital camera or photodetector.

Although the following detailed description makes often reference toimmunoassays chips, the detector and detection system of the presentdisclosure, can also be used in connection with microfluidic chipsadapted to perform DNA hybridization assays such as the ones performedin standard DNA microarrays, expression analysis of mRNA, certain formsof DNA detection and/or DNA sequencing (see e.g. Kartalov, Emil P. andQuake, Stephen R. (2004) Microfluidic device reads up to fourconsecutive base pairs in DNA sequencing-by-synthesis in Nucleic AcidsResearch, 32 (9). pp. 2873-2879 herein incorporated by reference in itsentirety).

In any of those embodiments, the test matrix can be expanded to measurea significantly large numbers of samples by exploiting the capabilitiesof microfluidic technology, resulting in even greater advantages ofparallel fluorescence measurement.

FIG. 1 shows a schematic representation of the device according to thepresent disclosure. A microfluidic circuit or chip 10 is located betweenan excitation filter 20 and an emission filter 30. The output image ofthe microfluidic chip 10 is detected by a combination of a lens 40 and acamera 50. In particular, lens 40 and camera or detector 50 will allow adigital image of microfluidic chip 10 to be obtained. The camera ordetector 50 can be, for example, a Canon EOS digital “Rebel” camera, aphotodiode, phototransistor or other detector. In order to excite thefluorescence source of the microfluidic chip 10, a light-emitting device60 is provided. The light-emitting device 60 can be a light emittingdiode (LED) or a mercury lamp or laser.

In an alternative embodiment, the excitation light source and detectorcan be placed at right angles to one another in a configuration thatuses a dichroic mirror in the standard configuration to increasebackground filtering and a Signal to Noise Ratio (SNR) (with referenceto use of SNR in the detector herein disclosed see below description ofFIGS. 5 to 7).

In certain embodiments, the detector of the present disclosure caninclude optical fibers, or bundles thereof, to deliver the excitationand emission light to and from specific locations on the chip, toparticular pixels or clusters of pixels on the detector. In particular,in those embodiments, the lenses in the detector can be replaced by theoptical fibers and the detector can include a CCD imager, a CMOS imager,an Avalanche Photodiodes (APD) array etc.

FIG. 2 shows a more detailed representation of the arrangement ofFIG. 1. In particular, the microfluidic circuit 10 includes afluorescence source 110 (e.g., the fluorophore Alexa Fluor 555 solutionin a concentration ranging, for example, from 0.01 g/ml to 300 g/ml inwater) associated with a microfluidic structure 120, both of which areon top of a glass piece 130. In some embodiments the microfluidic chip10 can be of the type shown in U.S. Ser. No. 11/439,288 “High ThroughputMulti-Antigen Microfluidic Fluorescence Immunoassays” filed on May 22,2006 and incorporated herein by reference in its entirety. Furthermicrofluidic chips, including but not limited to chips made ofelastomers or polymers such as PDMS, PMMA, Polyurethane, PFPE, SIFEL,parylene, and others, can be used in combination with a detector of thepresent disclosure. In some embodiments those chips are adapted toperform DNA hybridization assays, expression analysis of mRNA, certainforms of DNA detection and/or DNA sequencing. Those chips areidentifiable by a skilled person upon reading of the present disclosureand will not be further discussed here in details.

The light emitting device 60 is connected to a heat sink 70 which, inturn, receives cool air 80 from a fan 90. Both the light emitting device60 and the fan 90 are connected to power sources, not shown in thefigure. Although the heat sink 70 is optional, it is advantageous whenhigh power LED's are used as light emitting devices 60. In suchembodiments, the heat sink 60 gives better longevity and —moreimportantly—stability of the LED wavelength.

When combined with a LED source 60, the digital camera or detector 50provides a compact portable fluorescence imaging system, as two 6Vlantern batteries connected in series can be used to power theelectronics. The excitation filter 20 serves to protect the camera 50and can be, for example, HQ 545/25 by Chroma. The LED, laser diode ormercury light source 60 can be selected to emit at a wavelength ofapproximately 545 nm. A graph showing the emission and excitation curveof the exemplary fluorophores Alexa Fluor 555 is shown in FIG. 3.

In the following paragraphs preceding the discussion of FIG. 4A and FIG.4B, an exemplary technique for obtaining an exemplary circuit 10suitable to be used in combination with the detector of the presentdisclosure, will be explained.

PDMS microfluidic chips with integrated micromechanical valves werebuilt using soft lithography as described, for example, in “MonolithicMicrofabricated Valves and Pumps by Multilayer Soft Lithography”, byMarc A. Unger, Hou-Pu Chou, Todd Thorsen, Axel Scherer, Stephen R.Quake, Science 7 Apr. 2000, Vol. 288. no. 5463, pp. 113-116, and“High-throughput multi-antigen microfluidic fluorescence immunoassays byEmil P. Kartalov, Jiang F. Zhong, Axel Scherer, Stephen R. Quake, CliveR. Taylor, and W. French Anderson in Biotechniques 2006 Volume 40,Number 1: pp 85-90, both incorporated herein by reference in theirentirety, with the following modifications.

Silicon wafers were exposed to HMDS vapor for 3 min. Photoresist SPR220-7 was spun at 2,000 rpm for 60 sec on a Model WS-400A-6NPP/LITEspinner from Laurel Technologies Corp. The wafers were baked at 105 degC. for 90 sec on a hotplate. UV exposure through black-and-whitetransparency masks was performed for 1.75 min on a mask aligner (KarlSuss America Inc., Waterbury, Vt.). The molds were then developed for 2min in 100% 319 MicroChem developer. Flow layer molds were baked at 140deg C. for 15 min on a hotplate to melt and round the flow channels.Molds were characterized on an Alpha-Step 500 (KLA-Tencor, MountainView, Calif. 94043). Channel height was between 9 and 10 μm. Controlchannel profile was oblong, while flow channel profile was parabolic.Except for the height measurements, the mold fabrication was conductedin a class-10,000 clean room.

Molds were exposed to TMCS vapor for 3 min. PDMS in 5:1 and 20:1 ratioswere mixed and degassed using an HM-501 hybrid mixer and cups fromKeyence Corp. (Long Beach, Calif. 90802). Thirty-five grams of the 5:1was poured onto the control mold in a plastic petri dish wrapped withaluminum foil. Five grams of the 20:1 was spun over the flow mold at1,500 rpm for 60 sec on a Spincoater P6700 (Specialty Coating Systems,Indianapolis, Ind. 46278). Both were baked in an 80deg C. oven for 30min. The control layer was taken off its mold and cut into respectivechip pieces. Control line ports were punched using a 20-gauge luer-stubadapter (Beckton-Dickinson, Franklin Lakes, N.J. 07417). Control layerpieces were washed with ethanol, blown dry with filtered air ornitrogen, and aligned on top of the flow layer under a stereoscope. Theresult was baked in an 80 deg C. oven for one hour. Chip pieces werethen cut out and peeled off the flow layer mold. Flow line ports werepunched with a 20-gauge luer-stub adapter. Chip pieces were washed inethanol and blown dry before binding to the epoxide glass slides. Thenow assembled chips underwent final bake in an 80 deg C. oven overnight.

Sylgard PDMS elastomer was mixed with concentration (10:1), poured ontoa silicon/photoresist mold, and used in a replication molding procedure.Once PDMS channels were completed, holes were mechanically punched toaccess the PDMS channels, and the elastomer was bonded to glasssubstrates at 80 C. Following this soft lithography procedure, the glasswas covered with black electrical tape to avoid stray light penetration.The chip was ready to be used as described in in U.S. Ser. No.11/439,288 “High Throughput Multi-Antigen Microfluidic FluorescenceImmunoassays” filed on May 22, 2006 and incorporated herein by referencein its entirety.

FIG. 4A and FIG. 4B show images taken with the arrangement shown in FIG.1 and FIG. 2 following injection of a chip 10 with differentconcentration of the fluorophores Alexa Fluor 555 ranging from 0.01 to300g/ml in water. FIG. 4A shows a view of the microfluidic chip 10 with100 micron wide fluid channels taken with only the emission filter 30 inplace, in order to show the resolution of the device according to thepresent disclosure. FIG. 4B shows a 100 micron wide by 10 micron tallchannel filled with Alexa Fluor 555 in a fluorescent image from thedevice according to the present disclosure.

The intensity in the resulting digital image matches the concentrationof the Alexa dye—in the sense that only the fluorescent light 110matching the filter 20 reaches the chip 120—and follows a non-linearrelationship for higher dye concentrations as shown in the followingFIG. 5, where the Alexa Fluor 555 dye emits at 555 nm once excited.

In FIG. 5, signal to noise values of the fluorescence signal werenormalized by applicants and plotted as a function of concentration.This number was found to generally increase with concentration whenmeasuring the chip during UV lamp illumination. Above concentrations of0.1 mg/ml, a linear relationship between fluorescence signal/noise andconcentration is observed. At lower concentrations, theintensity/concentration graph reveals an anomalous peak in florescenceintensity at 0.030 mg/ml. This peak, which indicates that thefluorescence drops with increasing dye concentration, can be explainedby quenching mechanisms between dye molecules bonded to the surface ofthe flow channels and is also explained with reference to FIG. 6 andFIG. 7. If, instead of a mercury lamp, a LED (with a 2.5×largerintensity) at the desired excitation wavelength (xlamp 750 mW from cree)is employed as a fluorescence source, the SNR values are improved andthe fluorophore is found to saturate.

As shown in FIG. 5, four interesting regions can be identified in thefluorescence SNR graph from highest concentration to lowest (right toleft). In particular:

A—Saturation: At sufficiently high concentrations, saturation occurs inboth the volume of the liquid and at the surface of the flow channels asthe dye molecules are close enough to result in fluorescence quenching.Saturation on the surface results in significant fluorophore quenchingas the fluorophores are very closely spaced. As the concentration offluorophore is increased, non-specific sites can be driven towardsfluorophore attachment. At even higher concentrations, the CMOS detectorelements may saturate, leading to further nonlinearities in thefluorescence measurement. Moreover, at high concentrations, horizontalscattering of light out of the channel sides leads to a decrease in SNR.

B—First linear region: At lower concentrations, the CMOS detector arrayresponse becomes linear and volume quenching is largely avoided.However, surface Fluorescence Resonance Energy Transfer (FRET) energytransfer still occurs, but this term is expected to be linear withrespect to concentration.

C—First rise in graph: Above a critical concentration, the fluorophoredensity in the volume is sufficiently high to ensure that the surfacedye molecules are saturated and experience quenching. As theconcentration decreases, fluorophores molecules are released from thesurface layer and, as a result, dye molecules that were previouslyquenched now contribute to the fluorescence signal.

D—Second linear decrease: as the concentration is further decreased, thefluorescence signal from the liquid and the surface are linearly relatedas no quenching compromises the fluorescence signal. To avoiduncertainties with the nonlinear intensity behavior resulting fromfluorescence quenching, it is possible to bleach the fluorophores. Ifthe signal is reduced after optical irradiation, fluorophoreconcentrations are below the ones relevant for quenching. However, ifthe fluorescence intensity is increased, fluorescence quenchinginfluences the signal. During optical irradiation, both freefluorophores contributing to the fluorescence signal as well asfluorophores that quench neighboring molecules are bleached. In mostrealistic clinical cases, surface treatments that decrease nonspecificattachment to the surface and result in contributions from the surfacewill not be very important as the fluorescence dependence onconcentration is linear for dilute concentrations. However, non-specificattachment to a surface can be characterized by observing thefluorescence quenching.

Filters for other wavelengths (HQ 605/75, HQ 610/75, HQ620/60) were alsotested to obtain superior signal to noise values.

To improve the quality of the pictures and eliminate stray light, anopaque rectangular aperture can be placed above the chip 10. Inparticular, the dimensions of the aperture can be chosen to be slightlysmaller than those of the PDMS chip, to ensure that only filtered lightemission from the chip reaches the camera.

Digital images of the chip 10 can be taken, for example, at ISO settingsof 100, 400 and 1600 and with shutter speeds ranging from 1/100 secondsto 2 seconds. Image analysis software (e.g., Astra Image 2.0 from PhaseSpace Technology) can be used to analyze these pictures. The measuredred signal of the channel can be compared to the red signal from thebackground to evaluate the signal to noise using the above software. Thefollowing equation can be employed:${S/N} = \frac{{{Red\_ mean}{\_ channel}} - {{Red\_ mean}{\_ background}}}{\left( {{Red\_ deviation}{\_ background}} \right)({Shutter\_ speed})}$

One of the advantages of conducting an antibody assay is the highspecificity offered the antibodies bound to the epoxide surface,according to the epoxide chemistry to prepare the surface. After theanalyte containing the antigen to be identified is flowed over thatcarefully prepared surface, the antigen is selectively removed from theflow and concentrated on the antibody-treated regions of the flowchannels. Of course, the probability of binding can be increased byusing microfluidic channels that pump the solution over theantibody-coated surface many times. It is important to ensure that manymore binding sites are available than antigen molecules in the analyte,so that the fluorescence or absorption intensity can be quantitativelyrelated to the antigen concentration. In a fluorescence immunoassay, thefluorescence intensity can be obtained with a digital image. With theappropriate selection of antibody chemistries deposited on the surfaceof an immunoassay chip, and after careful calibration to a develop a“standard curve” which gives signal intensity verses concentration forthe system, a simple snapshot image suffices to provide analyticalinformation, both qualitative and quantitative about many antigenswithin a fluidic specimen. The concentration obtained by the antibodysurface treatment can lead to 1000× increases in the local concentrationand thereby the fluorescence intensity of the surface compared with thatof the liquid analyte.

In the table of FIG. 8, applicants have summarized some of theclinically relevant concentrations that need to be measured within bloodsamples for the particular application of cancer marker detection. Ascan be seen from the table of FIG. 8, concentrations ranging from the 10pg/mL-150 mg/dL need to be measured, compared to 100 μg/mL minimumsensitivity demonstrated with our present digital camera instrument.

Therefore, antigens have to be concentrated significantly before suchsmall concentrations can be measured, but this is enabled throughmicrofluidic delivery of large volumes of sample over the functionalizedsurface. This observation renders the microfluidic immuno-assay approachmuch more sensitive than typical micro-array technologies, in whichdiffusion must take place before antigens can react with thefunctionalized surface. Thus, in micro-arrays, either much higherconcentrations or longer times should be provided to obtain similarsensitivities limiting the use of simple digital cameras with limitedsignal to noise performance.

FIG. 8 also indicates the time required for a typical clinical analysisto be completed, and this ranges from several hours to several days. Itis naturally desirable to reduce the time required to obtain testresults and this is enabled through the use of inexpensive fluidics andimaging systems.

Applicants have shown that concentrations of 0.01-1000 mg/ml can bemeasured with a simple commodity off the shelf CCD camera well below theranges needed for clinical evaluations of blood samples in microfluidicchips. When possibly combined with fluorescent assays and finger prickto plasma technologies this represents the core of a cheap, accessiblemedical testing. In particular, the components shown in FIG. 1 and FIG.2 are all commodity off-the-shelf pieces, so that the sensitiveimmunoassay detector according to the present disclosure can be builtfor a few hundred dollars.

According to further embodiments, methods and devices, in particularmicrofluidic devices, for filtering fluids such as bodily fluids, aredisclosed. Devices and components for filtering bodily fluids and inparticular blood are known as such. Reference can be made, inparticular, to U.S. Ser. No. 11/297,651, “Prototyping Methods andDevices for Microfluidic Components”, filed on Dec. 7, 2005 andincorporated herein by reference in its entirety.

In the above application, the process of providing a “negative” mold,using wax, pouring a polymer on the mold to form the “positive” of thestructure, and finally melting away the wax is described in detail. Thefinal result of the process is a three-dimensional structure comprisinginterconnected microfluidic components comprising channels, vias andcontrol sections.

The present disclosure provides a particular embodiment of amicrofluidic filtering structure. More specifically, it provides amicrofluidic blood filtering structure. In particular, the microfluidicfiltering structure herein disclosed, comprises microfluidic channelswhich are completely sealed to a microfluidic filter, such that allfluid in the channel must pass through the filter. In particular, themicrifluidic channels size can is between 5 microns and 3 microns.

FIG. 9A shows a perspective view of the negative mold 610 used to form amicrofluidic filtering device. The negative mold 610 can, for example,be made of aluminum.

The mold 610 comprises a first or top pin 620 and a second or bottom pin630. The first pin 620 is a cylinder designed to mold the polymer (e.g.,PDMS) such that the capillary tubes connecting to the microfluidic bloodfilter can be pushed in and sealed. The second pin 630 comprises a 24gauge steel pin press fit into the aluminum in order to mold a holeduring the casting process to allow a tight seal to standard 23 gaugepins typically used for microfluidics. The person skilled in the artwill understand that diameters and materials can be changed according tothe various uses.

The top pin 620 and bottom pin 630 are designed to be separated so thata piece of filter material (shown in a later figure) can be placedbetween them. The top pin 620 is connected to a top piece 640, while thebottom pin 630 is connected to a bottom piece 650. The top piece 640 andthe bottom piece 650 are connected by screws 660, 670. The distancebetween top piece 640 and bottom piece 650 can be adjusted by means ofbolts 680, 690. Adjustment of the distance allows the filter element tobe compressed between the top piece 640 and the bottom piece 650. Thisensures that a leak tight channel will be created in which blood mustpass through the filter only. The top piece 640 also comprises holes700, 710 that can be threaded and used to gently back the mold out ofthe polymer microfluidic structure when the casting process is finished.

FIG. 9B shows a cast microfluidic polymer structure with an embeddedfilter element obtained through use of the mold of FIG. 9A in a castingprocess such as the one described in U.S. Ser. No. 11/297,651.

In particular, a cast structure 800 is shown, which comprises thepolymeric “positive” of mold 610 of FIG. 9A, together with an embeddedfilter element 810 located between the polymeric positive of top pin 620and the polymeric positive of bottom pin 630, which constitutes themicrofluidic channels in the structure.

During the process of fabrication to reach the structure of FIG. 9Bstarting from the mold of FIG. 9A, the filter paper 810 is placedbetween top pin 620 and bottom pin 630 and then bolts 680, 690 are usedto squeeze and hold the filter paper in place. In an alternativeembodiment the filter can be cast in a wax mold shaped like the metalmold of FIG. 9A with the filter trapped in the middle. A polymer such asPDMS or SIFEL can be poured over the mold and cured, and then the waxmelted out of the cured polymer, creating basically the same trappedfilter and microfluidic channels in a monolithic polymer piece. At theend of the casting process, a monolithic three-dimensional PDMS deviceis obtained.

In certain embodiments a plurality of microfluidic filters is formed bymultiple molds, such as the one described in FIG. 9A. In theseembodiments, forming and releasing of the mold can be performed at thesame time.

There are several commonly available filters, all of which are verycheap, especially in the small size needed for the device according tothis disclosure. Applicants have chosen the BTS-SP series from PallCorporation (East Hills, N.Y.). The BTS-SP medium features a highlyasymmetric membrane that is specifically engineered for serum separationof whole blood, shown in the micrograph of FIG. 10. The graduated porestructure of the filter includes more open pores 910 on the upstreamside and finer pores 920 on the downstream side. This high degree ofasymmetry allows red and white blood cells to be captured in the largerpores while the plasma wicks into the smaller pores on the downstreamside of the membrane. The large pore side of the medium serves as anabsolute cell exclusion zone and performs very well in the deviceaccording to the present disclosure.

In some embodiment filtering can in principle include additional methodsof purification or filtering, including but not limited to liquidchromatography, bead purification column, nanofabricated porous siliconmembranes, or any other known method of purification or filtration (seee.g. Menake E. Piyasena, Tione Buranda, Yang Wu, Jinman Huang, Larry A.Sklar, and Gabriel P. Lopez* “Near-Simultaneous and Real-Time Detectionof Multiple Analytes in Affinity Microcolumns” in Anal. Chem. 2004, 76,6266-6273, and L. Hernandezl, M. Rudolphl, R. Lammertink2, J.Kornfield2, C. Zurital and F. A. Gomezl, Determination of BindingConstants of Polyethylene Glycol Vancomycin Derivatives to PeptideLigands Using Affinity Capillary Electrophoresis in ChromatographiaVolume 65, Numbers 5-6/March, 2007, both incorporated herein byreference in their entirety). A person skilled in the art, upon readingof the present disclosure, would be able to identify additionalfiltering system to be used in conjunction with assembly that will notbe further discussed herein in details.

Turning back to the process of fabrication to reach the structure ofFIG. 9B starting from the mold of FIG. 9A, when the mold 610 has beenprepared, reference can be made to the flow chart shown in FIG. 11. Themold 610 is placed sideways into a standard container such as a Petridish (step S1). A polymer, such as PDMS is then poured into the Petridish (S2), and care is taken to completely cover the mold. The PDMS ismixed in a 10:1 ratio. After the mold is completely covered by the PDMSmixture, it is left to degas in vacuum (S3), until all air bubbles havebeen removed. At this point, the mold and the PDMS in the Petri dish areleft to cure in an 80 degree Celsius oven for 1 hour (S4). After curing,the PDMS blood filter is cut out of the dish (S5) and the bolts andscrews are removed from the mold (S6), freeing the two halfs. The boltsand screws are then inserted into the separation holes 700, 710 of FIG.9A (step S7) and the mold is slowly backed out of the PDMS (S8). Thisprocess allows the blood filter paper 810 shown in FIG. 9B to remainintact in the PDMS filter.

In accordance with the present disclosure, the polymeric structureincluding the filter can further be provided, with at one end aninputting or receiving channeling structure, and on another end anoutputting channeling structure.

In particular, the receiving channeling structure can be integrated witha capillary tube with an anti-coagulant (e.g.,ethylene-diamine-tetra-acetic acid, (EDTA)) on it. In particular, thereceiving end of the polymeric structure can be provided withcapillaries of the type that are commonly used for finger stick blooddraws and are available with a variety of anti-coagulants depending onthe intended analysis to be performed. In some embodiments,anticoagulents can also be applied to the microfluidic channels formedby the polymeric positive of top pin 620 and bottom pin 630. Applicationcan be performed for example via spray-dry coating or other method, suchas evaporation of anticoagulant solutions in the microfluidic channels.As a consequence incoming blood can be anticoagulated in themicrofluidic filter without relying on the capillary tube to add theanticoagulant.

The outputting other end of the blood filter, the end through whichfiltrate passes through, was made to interface with any standardmicrofluidic chip, e.g., through a 23 gauge pin. Whole blood would passthrough the specially designed filter paper embedded in the PDMS bloodfilter, separating whole blood from the plasma, which would be used inthe microfluidic chip.

The device according to the present disclosure can use the standardmethod of a finger stick blood draw using a capillary tube to providethe microfluidic circuit described above with blood plasma or serum thatis ideally suited for downstream microfluidic evaluation.

The choice of the capillary tube to bring blood to the microfluidicfilter in accordance with the present disclosure is specific to the testto be carried out on the blood because different anticoagulants areneeded for different analyses. By way of example, the applicants haveused the capillary tube StatSampler Capillary Blood Collectors fromStatSpin (Iris Sample Processing, Westwood, Mass.) which has EDTA as ananti-coagulant to keep the blood sample from drying out or otherwisebecoming unusable. These capillary tubes are used for example indoctor's offices as a standard finger prick blood draw and are availablewith several different anticoagulants. The person skilled in the artwill understand that different capillary tubes and differentanticoagulants can be used in conjunction with the microfluidic filterin accordance with the present disclosure.

In some embodiments the fluid is introduced through a pipe or a metalconnector. In some embodiment the microfluidic channels can be coatedwith anticoagulant, for example by applying spray-dry method, or byfilling the channel with an anticoagulant solution and allowing itevaporate (either naturally or by heating, or also by lyophilization)

Moreover, in the specific experiment conducted by applicants, a mouseblood sample from Bioreclamation Inc., already containing EDTA, wasused, thus rendering the presence of an anticoagulant inside thecapillary tube not necessary. In particular, 20 ml of mouse blood wasstored and refrigerated at 4 degrees Celsius when not used, comprisingtwo 10 ml samples, drawn a week from each other. The mouse blood wasthen drawn, 1 ml at a time, into a capillary tube. The capillary tubewas then inserted into the upper portion of the filter.

In certain embodiments, the fluid can be drawn into the inputtingmicrofluidic channel, either by capillary action or by vacuum and it ispassed through the filter into the outputting microfluidic channel. Insome of those embodiments, wherein a positive pressure is applied a capcan be applied to the inputting end of the microfluidic filtering deviceto seal it and apply pressure through the microfluidic channels thefluid is introduced into said microfluidic channels. In some of thoseembodiments the inputting end of the device/structure is shaped like acone to ease introduction of the fluid.

FIG. 12 shows a perspective view of the PDMS blood filter of the presentdisclosure integrated with a microfluidic blood analysis chip and ablood draw capillary tube. In particular, the polymeric blood filteringstructure 500 comprises a hollow cylinder 540 (which is a positive castof the upper aluminum pin 620 of the mold 610) and a hollow pin 530(which is a positive cast of the lower aluminum pin 630 of the mold610), together with a blood filter 550 (e.g., the paper filter describedin the example above). The filtering structure 500 is connected to acapillary tube 510 on it upper side and to a blood analysis microfluidicchip 520 on its lower side. It can be seen that the hollow pin 530 isconnected to the blood filter 550 on its top side and to themicrofluidic chip 520 on its bottom side.

The capillary tube 510 can be attached to a hose (not shown) suppliedwith dry nitrogen and the blood pumped through the filtering structure500 at a constant pressure of 0.5 psi. This pressure is chosen byapplicants for safety an d to demonstrate that very little pressure isnecessary to flow blood through the filter. The pressure by which bloodis filtered can also be stabilized. Increasing the pressure from theabove low level does serve to speed the blood flow. However, 1 ml ofblood at a pressure of 0.5 psi went through the filtering structure 500quickly. The filter 550 was shown to collect 80-100% of the availableblood plasma which is typically half the total volume of the blood. 500microliters of blood plasma is a very large amount of blood for mostmicrofluidic applications which typically require nanoliters tomicroliters of fluid in order to perform analysis. A person skilled inthe art would understand that additional pressure can be used as well asthat a vacuum that can be applied to suck the fluid through the filterand the microfluidic chip

FIG. 13 shows a simulated side view of the blood filter device duringoperation. The blood cells are all kept above the filter and plasmapasses through below. In particular, the blood cells are stopped byfilter material 420 and plasma continues to flow past the filter. Oneadvantage of this design is that the same pressure or vacuum whichpushes or pulls the blood through the filter can also be used to pushthe blood through the attached microfluidic chip 520 of FIG. 12.

FIG. 14 shows two microfluidic channels, one with anti-coagulated mouseblood flowing through it and the other with the filter directlyconnected to its input port. In particular, both channels 430, 440 havedimensions of 100 micrometers in width, channel 430 with anti-coagulatedmouse blood flowing through it and channel 440 with the filter directlyconnected to its input port. Channel 440 is full of blood cells, whilechannel 430 is clear and is flowing only plasma.

In summary, according to one of the aspects of the present disclosure, aPDMS microfluidic blood filter has been disclosed. The filter can beused with on-chip whole blood filtration for microfluidic bloodanalysis. The filter is able to filter the necessary volume of blood inan acceptable time frame. The PDMS blood filter can be used withstandard microfluidic chips and is effective in separating plasma andblood cells from whole blood. The ability to collect whole blood from asimple finger prick and directly insert it into a microfluidic chip willallow blood analysis to be brought closer to the patient and eliminatethe need for a painful venipuncture and a trained phlebotomist andsimplify the collection and analysis of blood.

In some of the embodiments of the present disclosure, poly(dimethylsiloxane), or PDMS, was the material used for the blood filter,as it is more cheap and disposable, and has little affinity forproteins, RNA, DNA etc. The device can also be made from a variety ofpolymers including fluorinated Sifel and PFPE if the specificapplication requires the highest capture rate and sensitivy for rarespecies. PDMS is a good choice because it seals well to the filter, iscompatible with traditional microfluidics and is flexible enough to sealto both the glass capillary tube and the standard metal pins used tointroduce samples to microfluidic chips.

The fluorescence assay detector and the microfluidic filterdevice/polymeric structure, described above are combined in amicrofluidic filtering and detecting system/assembly, which can be usedfor example for diagnostic purpose.

In the microfluidic assembly, the microfabricated filter can have anyspatial orientation, including orientations that are sideways in theplane wherein the microfluidic chip is located.

In some embodiments, the microfabricated filter can be in principleproduced, and desirably so, as a monolithic part of the microfluidicdevice itself, e.g. by being imbedded in the thick upper layer of thechip, during chip fabrication.

The system can also include a concentration stage/device whereby thefluid to be tested, for example urine, is processed through a capturesystem. The capture system can include but not limited to immunobeads orDNA hybridization beads, which would allow the processing of largequantities of sample for the detection of a molecule that, in view ofthe limited concentration in the fluid, would otherwise be difficult orimpossible to handle in microfluidic devices within reasonable operationtime scales (see e.g. Menake E. Piyasena, Tione Buranda, Yang Wu, JinmanHuang, Larry A. Sklar, and Gabriel P. Lopez* “Near-Simultaneous andReal-Time Detection of Multiple Analytes in Affinity Microcolumns” inAnal. Chem. 2004, 76, 6266-6273 incorporated by reference in itsentirety).

In principle, the system may also include one or more additionalsubsystems/subassemblies e,g, for the performance of stages such as DNAsequencing PCR and/or RT-PCR in the sample (see e.g. Kartalov, Emil P.and Quake, Stephen R. (2004) Microfluidic device reads up to fourconsecutive base pairs in DNA sequencing-by-synthesis. Nucleic AcidsResearch, 32 (9). pp. 2873-2879, herein incorporated by reference in itsentirety). In principle, the one or more subsystems can be operated inthe assembly at a stage that precedes or follows the detection of themolecule of interest in the micro chips. A person skilled in the art,upon reading of the present disclosure will be able to identify thosesubsystems/subassemblies which will not be further described herein indetails.

In certain embodiments, the microfluidic filtering and detectingassembly is arranged as a portable diagnostic system/assembly. In thoseembodiments, the diagnostic system/assembly comprises: a samplepreparation stage/component: and a sample analysis stage/component, todetect analytes. The sample preparation stage/component can comprise amicrofluidic circuit with a fluorescent fluid source, and the sampleanalysis stage/component can comprise: an excitation filter; an emissionfilter; a light emitting device to excite the fluorescent fluid source;and a detecting arrangement for detecting fluorescence images from themicrofluidic circuit, with the microfluidic circuit located between theexcitation filter and the emission filter.

In particular in the portable diagnostic system/assembly, the sampleanalysis stage is for the detection of important analytes at clinicallyrelevant levels, wherein the detection is performed according to methodsand devices apparent to the skilled person upon reading of the presentdisclosure. In an exemplary embodiment a portablefluorescence-immunoassay platform is used to measure many analytes onthe same circuit at the same time, with indigenous sample preparation,and at clinically relevant levels.

In certain embodiments, a computer circuit is also included thatcontrols a vacuum system (or positive pressure pump) to move the fluid,in particular plasma, through the filter and the chip in a controlledfashion. In some embodiments the computer also controls the LED, thedetector, and gives feedback to patient and also to the doctor (e.g. viainternet), stores data, gives patient history in graphs etc.

An advantage of the portable diagnostic system herein disclosed is thatallows patients to obtain a diagnostic indication, by performing thefollowing operations, introducing a microfluidic apparatus in thediagnostic assembly, or simply using another test in a multi usedisposable chip, introducing the fluid to be tested, for example in caseof blood analysis by puncturing a finger an place the finger over aninputting microchannel structure and reading the diagnostic indicationprovided, for example in a visual display.

In the following paragraphs, two different applications of the abovediscussed devices methods and systems will be shown. While the twoapplications below deal with the use of the system described above insmall cell lung carcinoma and in multiple sclerosis, the person skilledin the art will understand that many other applications are possible.

A. Use in Small Cell Lung Carcinoma

In the following paragraphs, the application of a microfluidicfluorescent noncompetitive immunoassay system as the one described abovewill be discussed for the detection of the CRMP5 protein, a marker forSCLC (Small Cell Lung Carcinoma), and the miniaturization of thedetector. Using monoclonal rat antibodies, an immunoassay stack specificto the CRMP5 marker was built and tested for specificity andsensitivity. A microfluidic filter device of the present application wasused to able to separate serum from blood allowed for tests to examinemarked blood. In particular, the filter device shown in FIG. 9B of thepresent application was used. A detector as shown in FIG. 2 of thepresent application, comprising an excitation and emission filter set,lens, LED, and CCD digital camera replaces bulkier optical microscopedetectors and allows for images to be taken simultaneously off allchambers in the microfluidic device. By achieving appropriate signal tonoise ratios with the miniaturized detector of FIG. 2 and using theimmunoassay chip like the one described in U.S. Ser. No. 11/439,288 intandem with microfluidic blood filters like the one described in FIG. 9Bof the present application, the Applicants approach cost efficienthandheld devices capable of detecting SCLC with high specificity andsensitivity.

Using a two-layered PDMS chip for microfluidic fluorescence immunoassayas a platform (see, e.g. U.S. Ser. No. 11/439,288), Applicantsinvestigate the creation of a point of care testing device capable ofdetecting the presence of the antigen CRMP5, a protein marker forsmall-cell lung carcinoma (SCLC) and in some cases thymoma. Applicantsconducted a sandwich immunoassay test on a sample containing CRMP5 forproof of concept testing on the device's ability to detect a specificantigen. Collected data was analyzed for specificity and sensitivity. Aminiaturized fluorescence detector was constructed from an inexpensiveCCD camera, LED, lens and emission and excitation filter as shown inFIG. 2 above. A chip with fluorescence signal was observed through thissystem to show such a device was capable of simultaneously inspectingall chambers and still retain appropriate signal to noise ratio.

With the microfluidic chip sandwich (see element 10 of FIG. 2),immunoassays have been performed on a microscopic level and at antigenconcentrations as low as 10 picoMolar and saturation concentrations inthe 100 nanoMolar range. Here, Applicants conducted a proof of conceptexperiment; using the chip to detect the CRMP5 antigen, a marker ofsmall-cell lung carcinoma, in the saturation range. In order to simplifythe process fluorescently pre-tagged antibodies were used instead ofbiotinylation or tagged streptavidin. In order to investigate thepracticality of a point-of-care testing apparatus constructed aroundsuch a platform, a portable and low cost detector like the one shown inFIG. 2 was developed and tested.

The materials and fabrication instructions for the manufacturing thechips can be seen, for example, in U.S. Ser. No. 11/439,288. The chipconsisted of a control (top) layer and flow (lower) layer. The flowlayer contained a 5 by 10 matrix of pathways 10 micrometer tall and 100micrometer wide.

IgG rat monoclonal antibodies in ascites fluid were obtained from theMayo Clinic in three variations. CR-1 binds to residues ˜369-564 ofCRMP-5, CR-3 binds to residues ˜1-64, and CR-5 binds to residues˜57-376. CRMP-5 antigen, a 62 kilaDalton molecule, prepared from E. Coliin a 4.7 mg/mL concentration in dilute Phosphate Buffered Saline (PBS)was obtained from the Mayo Clinic.

Samples of each antibody were tagged with DyLight 547 using the PierceProtein Labeling kit supplied by Pierce Biotechnology (Rockford, Ill.,USA). The dye excites at 557 nm and emits at 570 nm.

PBS from Irvine Scientific (located Santa Ana, Calif., USA) and bovineserum albumin purchased from Sigma (St Louis, Mo., USA) were used tocreate the solution buffers. Tagged antibodies and the CRMP-5 antigenwere reconstituted in PBS 0.1% BSA solution. Untagged antibodies werereconstituted in pure PBS 1× solution. Trishydroxymethylaminomethane(Tris Buffer) from Sigma-Aldrich (St. Louis, Mo., USA) was used as aflushing and pacifying agent.

An Olympus IX71 inverted microscope (Olympus America Inc. Melville,N.Y., USA) equipped with mercury lamp, Texas Red emission and excitationfilter set (Absorption wavelength 595 nm, Emission wavelength 620 nm),and DFW-V500 Digital CCD cool charged-coupled device (CCD) from Sonyserved as the primary means of detection.

A miniaturized detector consisting of a 520nm-535nm (Green) Cree XLamp 37090 LED from ETG Corp (Los Angeles, Calif., USA) (see element 60 inFIG. 1 and FIG. 2), an emission and excitation filter set from Chroma(Excitation: HQ535/50x Emission: HQ610/75M; Rockingham, Vt., USA)—seeelements 20 and 30 in FIG. 1 and FIG. 2—, and a Canon EOS digital Rebelcamera (see element 50 in FIG. 1 and FIG. 2), was constructed andtested. Astra Image 2.0 software (Phase Space Technology) analyzedfluorescent signal captured from both systems.

The microfluidic chip was mounted upon Super Epoxy SME glass slides fromTeleChem International, Inc. (Sunnyvale, Calif., USA) as described inKartalov et al. in Biotechniques 2006 number 1 volume 40, pp 85-90,herein incorporated by reference in its entirety. Applicants plugged 23gauged tubes from New England Small Tubes (Litchfeld, N.H., USA) intothe control and flow input ports and used ID: 0.020 inch, OD: 0.060 inchTygon Tubing from Cole-Parmer (Vernon Hills, Ill., USA) to connect thecontrol channels to Lee-valve arrays (Fluidigm; San Francisco, Calif.,USA) and the flow channels to the reagent supply. The related unfilteredimage and schematic diagram of the microchip are shown in FIGS. 15A and15B respectively.

Pressurized water filled the control layers until all air had diffusedout of the channels. Applying pressure to any control input port via thesoftware controlled valve arrays closed a microfluidic valve and shutoff the flow in the corresponding reagent channel. Untagged monoclonalantibodies (CR5) were sent into the A5 input port and allowed to flowdirectly to the Test Output ports for several minutes. The horizontalflow channels were then flushed with Tris Buffer from the Test BufferInput port to pacify any free epoxide locations and remove excessantibodies from the channel. Next, two samples were sent through thefirst and second Sample Input Ports. The first solution consisted of 5%CRMP5 by volume in a PBS 0.1% BSA mixture, the second a controlcontained only PBS 0.1% BSA. The vertical channels were filled and thencirculated clockwise with a series of peristaltic pumps with a lap timeof 20 seconds for 10 laps.

This process was repeated 10 times with the vertical channels beingreplenished after each cycle in order to ensure the antigens bonded tothe antibody locations thoroughly. The vertical channels were thenflushed with Tris Buffer from the Sample Buffer Input to the SampleExhaust to remove the sample solutions. Fluorescently tagged monoclonalantibodies (CR1) were sent through the D5 input port and allowed to flowinto the Test Output port for several minutes, completing the stack.Tris Buffer was sent through the Test Buffer Input to remove standingfluorescence.

The chip was placed upon the stage of the Olympus inverted microscope,excited by the mercury lamp, and examined through the Texas Redemission/excitation filter cube. Using a simple CCD camera images weretaken using the microscope setup of the chambers containing both theantigen present sample and the control sample, and examined with AstraImage 2.0 software to determine the signal to noise ratio.

In order to determine the quality of signal capture from theminiaturized detector in comparison to the bulkier microscope setup, achip with a chamber of saturated signal was illuminated by the Green ETGLED through the HQ535/50x excitation filter and the signal captured bythe EOS digital Rebel camera through the HQ6 1 0/75M emission filter.The image was likewise analyzed with the Astra Image 2.0 Software todetermine the strength of the signal to noise ratio. The related signalsas viewed from a microscope detector and from the detector of thepresent disclosure are shown in FIGS. 16A and 16B respectively.

Using the above mentioned experimental approach, the images of the CRMP5present chamber and CRMP5 absent chambers were analyzed using AstraImage 2.0 software. The mean red signal of the chamber was compared withthe mean red signal of the background. The signal to noise ration (SN)was determined by dividing the net signal by the standard deviation ofthe background noise. Using the microscope setup a SN of 107 was foundfrom the CRMP5 positive chamber and a SN of 0.86 resulted from the CRMP5negative chamber, showing a high level of specificity.

Results from the miniaturized detector proved practical though lessproficient. Images taken from this portable setup of chambers saturatedwith fluorescence yielded a SN of 53.4. However the miniaturizeddetector proved capable of examining multiple chambers simultaneouslyand provided adequate qualitative data capable of differentiatingbetween antigen present chambers and antigen absent chambers.Additionally the detector proved able to produce images of the chip withhigh clarity and good focus while requiring less space and costlymaterials than the microscope detection system.

In view of the above, Applicants have shown the ability to detect theCRMP5 antigen with the microfluidic fluorescent immunoassays.Furthermore the above test demonstrates that tagging antibodies directlywith a protein dye proves as effective as attaching fluorescentstreptavidin without the need for biotinylated antibodies andsimplifying the immunoassay process. Due to time constraints and theshelf life of the proteins used, the sensitivity of the system withregards to CRMP5 has yet to be fully explored. Previous tests of thesystem with other antigens showed reliable detection as low as 10 pM.

B. Use in Multiple Sclerosis

The technology shown in the present application is also applicable fordetection and diagnosis of multiple sclerosis (MS), a debilitatingauto-immune disease. Applicants were able to demonstrate diseasedetection by identifying matrix metalloproteinase 9 (MMP-9), and GalCbiomarkers for MS, in simulated patient serum. This is aproof-of-concept for the viability of a handheld multiple sclerosisattack early warning system. Such a detector could prove invaluable inthe treatment of this disease. Multiple sclerosis is a disease thatprogresses through a series of exacerbations. If these exacerbations canbe predicted through constant monitoring of blood species, treatmentscan be developed for use at the onset of MS attacks in order to minimizeor eliminate the damage caused by the attack.

Multiple sclerosis is a debilitating auto-immune disease that affectsthe function of the central nervous system (CNS). An antibody responsetargets oligodendrocytes cells that are responsible for the myelinationof neural axons. The immune response critically damagesoligodendrocytes, halting production of myelin and leading to the rapiddemyelination and eventual disintegration of the axons. Ultimately, theaffected neurons lose the ability to conduct electrical impulses.

Diagnosis of multiple sclerosis is done through characterization of thesymptomatic response, which varies with each patient. More commonsymptoms, associated with the loss of neural function, include fatigue,numbness, spasticity, vision loss, and depression. Most patients fallinto one of the four identified symptom motifs for multiple sclerosis:relapsing-remitting (RR), primary-progressive, secondary-progressive,and progressive relapsing. A graph illustrating multiple sclerosissymptoms motifs is shown in FIG. 17.

Approximately 85% of multiple sclerosis patients are initially diagnosedas relapsing-remitting; characteristics of these patients includeepisodes of acute symptoms followed by remissions of partial to completerecovery. Within ten years of the initial diagnosis about 50% of RRpatients go on to exhibit secondary-progressive multiple sclerosis,which is characterized by a steady increase in symptom intensity withbrief periods of acute symptoms and recovery. A smaller percentage ofpatients, diagnosed as primary-progressive, exhibit a steady increase insymptom intensity from the onset of the disease. However, a very smallpercentage of patients with multiple sclerosis demonstrate a progressiverelapsing syndrome, wherein symptom intensity steadily increases alongwith intermittent periods of partial recovery.

Currently, there is no definitive serological test for multiplesclerosis. However, recent research has demonstrated the existence ofspecific biological markers for multiple sclerosis, which include matrixmetalloproteinase 9 (MMP-9), a gelatinase enzyme that has the ability tocleave myelin, and Galactocerebroside (GalC), a transmembraneglycoprotein in oligodendrocyte cells that is a known target fordemyelinating antibody responses. Furthermore, these markers have ademonstrated presence in the blood stream, making them promisingcandidates for immunological testing.

Applicants built a simple two-layer immunostack, using a high-throughputmicrofluidic immunoassay chip like the one described in U.S. Ser. No.11/439,288 also schematically shown in FIG. 18, to illustrate aproof-of-concept of multiple sclerosis disease detection. A serum ofMMP-9 was flowed through the coliseums of the fluidic chip and allowedto bond to free epoxide sites along the flow layer. Reference can bemade, for example, to the microfluidic chip shown in FIG. 2 of U.S. Ser.No. 11/439,288. After passivating the remaining epoxide sites with TRISbuffer, fluorescently-tagged monoclonal anti-MMP-9 antibodies werepumped throughout the coliseums. Subsequently, the coliseums wereflushed with TRIS buffer to liberate and remove and free-floatingantibodies. Finally, the fluidic chip was illuminated with light ofwavelength 547 nm, and the fluorescence emission was captured by a CCD.The results are shown in FIG. 19. Applicants inferred the presence ofMMP-9 because of the luminescence of the fluorophores.

Controlled detection of MMP-9 was done by fabricating a selective,three-layer protein stack (see FIG. 20) for MMP-9. First, polyclonalgoat anti-human MMP-9 molecules were flowed through the coliseums andallowed to bond to the epoxide layer of the immunoassay chip. Afterpassivating the remaining epoxide sides with TRIS buffer, simulatedpatient serum (consisting of a 470 nM solution of MMP-9) was flowedthrough the coliseums. The MMP-9 was immediately captured by thepolyclonal anti-MMP-9 antibodies. Finally, fluorescently taggedmonoclonal anti-MMP-9 antibodies were flowed through and mated with theMMP-9. Fluorescence imaging confirmed the presence of MMP-9 (see FIG.21). Furthermore, fluorescence was observed only in coliseums which hadexposure to MMP-9, validating the selectivity of the protein-stack.

Since only fully formed protein stacks have fluorescently taggedantibodies, the intensity of the fluorescence emission corresponds tothe strength of the signal. In one assay, Applicants measured the redmean of the signal to be 227 with a standard deviation of 39.2. Incomparison, the red mean of the noise was 9.224 with a standarddeviation of 9.23. This resulted an SNR of 24 to 1. In another assay,Applicants observed a red mean of the signal to be 72.6 with a noisefloor of 7.01, resulting in a SNR of 10 to 1. Successful immunoassaysconsistently had signal to noise ratios of 10 to 1 or greater.

In view of the above, Applicants demonstrated a proof-of-concept ofmultiple sclerosis disease detection through successful detection of thebiomarker MMP-9. Furthermore, fluorescence detection of MMP-9 yieldedSNR in excess of 24 to 1, with a minimum SNR of 10 to 1. Subsequenttesting has suggested that MMP-9 antigen detection is possible withconcentrations as low as 65 nM.

A high throughput immunoassay chip (see, e.g., element 10 in FIG. 1 andFIG. 2) was used to perform the microfluidic immunoassays. Softlithography was used to fabricate the chips in polydimethylsiloxane(PDMS), an organic elastomer. The PDMS chips were then mounted andbonded on SuperEpoxy SME glass slides from TeleChem International. (seechip shown in FIG. 22)

The immunoassays were carried out using human MMP-3, MMP-9 antigens,anti-MMP-9 polyclonal antibodies, and monoclonal human anti-MMP-9 andanti-MMP-3 antibodies manufactured by R&D Systems. Subsequentimmunoassays were performed with human GalC and anti-GalC antibodiesprovided by a collaborator.

The MMP proteins were reconstituted with TCNB buffer solution containing50 mM Tris buffer, 10 mM CaCl₂, 150 mM NaCl, and Brij 35 (a stabilizingdetergent manufactured by VWR Scientific.) The reconstituted proteinswere aliquot into single-use centrifuged tubes, and refrigerated at −27°C. until needed. Furthermore, monoclonal human anti-MMP-9 and anti-MMP-3were tagged with a Dylight 547 protein fluorescence labeling kit fromthe Pierce Corporation. The antibodies were stabilized with pure PBSbuffer from Irvine Scientific.

The actual immunoassay was controlled with a Fluidigm BOB3 pressure-flowcontroller and was interfaced to the user with the Fluidigm uChipsoftware. Antigen and antibody solutions were gently vortexed beforeinput. Tris buffer was used to passivate epoxide groups in reagentchannels. Excitation of the fluorophores was performed with a greenlight of 552 nm. Pictorial data was obtained using a Sony DFW-V500Fire-i digital camera.

In summary, Applicants have developed an inexpensive and rapid read-outsystem for multi-antigen microfluidic fluorescence immunoassay systems.The fluidic analysis chips can be fabricated in polydimethylsiloxanePDMS, and have been shown to perform high throughput analysis onmultiple analytes, with a measurement of over 100 fluorescent readingswith sensitive detection down to 0.01 mg/ml concentrations.

Applicants have shown a device that will allow a patient to have an athome testing device for daily monitoring of blood protein levels. Forinstance, in multiple sclerosis it has been very difficult to detect theearly stages of an attack in time to administer immunosuppressants in atimely manner in order to prevent nerve system damage. By usingmicrofluidic technology combined with inexpensive CCD or other detectorsit is possible to have an immunoassay chip as described in U.S. patentapplication Ser. No. 11/439,288 coupled with the CCD detector andappropriate electronics to monitor daily the levels of suspect proteinsor other species in the blood, e.g. from a simple fingerprick that ispassed through the chip. In this way the patient can be alerted when theproper marker protein rises so that he may alert his doctor and theappropriate action can be taken. Additionally, this chip can be used forresearching and finding such marker by comparing daily protein levelswith patient symptoms, outcomes and MRI scans.

Additionally, the above described system and method can be used forother types of diseases, such as a person with a known risk of cancer,or a patient who had hepatitis or some other known risk for certaintypes of cancer. Those persons can use a customized chip like the onedescribed above to search daily for cancer markers circulating in theblood. In this way the patient can spot the tumor months earlier than itotherwise would have been detected and patient outcomes can be improved.

While several embodiments of the invention have been shown and describedin the above description, numerous variations and alternativeembodiments will occur to those skilled in the art. Such variations andalternative embodiments are contemplated, and can be made withoutdeparting from the scope of the, invention as defined in the appendedclaims.

1. An microfluidic fluorescence detector comprising: an excitationfilter; an emission filter; a microfluidic circuit located between theexcitation filter and the emission filter, the microfluidic circuitcomprising a fluorescent source; a light emitting device to excite thefluorescent fluid source; a detecting arrangement for detectingfluorescence images on the microfluidic circuit.
 2. The detector ofclaim 1, wherein said detecting arrangement comprises a lens and acamera.
 3. The detector of claim 2, wherein the camera is a digitalcamera.
 4. The detector of claim 1, said microfluidic detector being aportable microfluidic fluorescence detector.
 5. The detector of claim 1,wherein the light emitting device is selected from the group consistingof a light emitting diode (LED), a laser diode and a mercury lamp. 6.The detector of claim 1, wherein the light emitting device is connectedto a heat sink.
 7. The detector of claim 1, wherein the light emittingdevice emits at a wavelength included in the entire visible spectrum. 8.The detector of claim 1, wherein the microfluidic circuit is a PDMSmicrofluidic circuit.
 9. The detector of claim 1, wherein thefluorescent source is Alexa Fluor
 555. 10. The detector of claim 1,wherein fluorescence images are detected in accordance with afluorescence intensity variation.
 11. The detector of claim 10, whereinthe fluorescence intensity variation allows four distinct regions to beidentified.
 12. The detector of claim 11, wherein the fluorescenceimages are refined in accordance with the fluorescence intensityvariation, by quenching the fluorescence source, and identifying aregion of the four distinct regions in the fluorescence variation, theregion associated with an image resulting from the fluorescence sourceonce quenched.
 13. The detector of claim 10, wherein the fluorescencevariation is calibrated to a standard curve to provide a quantitativeindication associated to a detected fluorescence image.
 14. The detectorof claim 1, wherein the detecting arrangement comprises one or more of:a CCD imager, a CMOS imager, a line scanner, at least one photodiode, atleast one phototransistor, at least one photomultiplier tube, at leastone avalanche photodiode.
 15. The detector of claim 14, wherein thedetecting arrangement further comprises at least one lens.
 16. A methodfor detecting fluorescence images, comprising: providing excitationlight at an excitation frequency to excite a fluorescence source,allowing the fluorescent source to emit light in connection with amicrofluidic apparatus; filtering the excitation light; filtering lightemitted through the fluorescent source; and detecting fluorescenceimages thus generated.
 17. The method of claim 16, wherein saiddetecting comprises digitally detecting fluorescence images thusgenerated.
 18. The method of claim 17, wherein said digitally detectingis taken at different settings.
 19. The method of claim 18, wherein saidsettings are ISO settings between 24 and
 3200. 20. The method fordetecting fluorescence images, of claim 16 wherein said method is fordetecting antigens in parallel.
 21. The method of claim 20, wherein oneof said antigens is antigen CRMP-5.
 22. The method for detectingfluorescence images of claim 16 wherein said method is for detectingbiomarkers in parallel.
 23. The method of claim 22, wherein one of saidbiomarkers is MMP-9.
 24. The method of claim 22, wherein one of saidbiomarkers is GalC.
 25. A microfluidic filter forming mold, comprising:a top pin; a bottom pin connected with the top pin but separable fromthe top pin, the bottom pin including an internal pin; a top piece withwhich the top pin is connected; and a bottom piece with which the bottompin is connected.
 26. The mold of claim 25, wherein distance between thetop piece and the bottom piece is adjustable.
 27. The mold of claim 26,wherein adjustment of the distance between the top piece and the bottompiece occurs through a pair of screws located between the top piece andthe bottom piece.
 28. The mold of claim 25, wherein the top piececomprises extraction holes.
 29. The mold of claim 25, said mold beingmade of aluminum.
 30. The mold of claim 25, said internal pin being madeof steel or plastic.
 31. The mold of claim 25, said mold comprising afilter located between the top pin and the bottom pin.
 32. Athree-dimensional polymeric structure comprising a polymeric cast of themold of claim
 25. 33. The three-dimensional structure of claim 32,further comprising an embedded filter element located between a cast ofthe top pin and a cast of the bottom pin.
 34. The three-dimensionalstructure of claim 32, the polymeric material being PDMS or SIFEL. 35.The three-dimensional structure of claim 32, said structure beingcoupled with a capillary tube input.
 36. The three-dimensional structureof claim 32, said structure being coupled with a hollow pin output. 37.The three-dimensional structure of claim 36, wherein the hollow pinoutput is connected with a microfluidic circuit.
 38. A microfluidicfiltering device, comprising a microfluidic filter cast in a singlepiece polymeric structure, the microfluidic filter connected to aninputting end of the structure through a microfluidic inputting channel,the microfluidic filter connected to an outputting end of the polymericpiece structure through an outputting microfluidic channel.
 39. Themicrofluidic filtering device of claim 38, wherein the device furthercomprises microfluidic channeling structures associated to the inputtingend and/or the outputting end of the structure.
 40. The microfluidicfiltering device of claim 38, wherein the microfluidic channels arecoated with an anticoagulant
 41. A microfluidic filtering and detectingassembly comprising: the three-dimensional structure of claim 32; andthe detector of claim
 1. 42. A process of fabricating athree-dimensional filtering structure, comprising: providing a moldhaving a top pin, a bottom pin and a filter between the top pin and thebottom pin; pouring polymer on the mold; curing the mold with pouredpolymer; separating the top pin from the bottom pin to free the filter;and extracting the mold from the polymer while leaving the filter in thepolymer.
 43. The process of claim 42, wherein the mold is placed in aconatiner before polymer is poured on the mold.
 44. The process of claim43, wherein the polymer is PDMS.
 45. The process of claim 42, whereinseparation of the top pin from the bottom pin occurs by removal ofscrews included in the mold.
 46. The process of claim 45, whereinextraction of the mold from the polymer occurs by reinsertion of thescrews in the mold.
 47. A portable diagnostic system comprising: asample preparation stage: and a sample analysis stage to detectanalytes.
 48. The portable diagnostic system of claim 47, wherein thesample preparation stage comprises a microfluidic circuit with afluorescent fluid source, and the sample analysis stage comprises: anexcitation filter; an emission filter; a light emitting device to excitethe fluorescent fluid source; and a detecting arrangement for detectingfluorescence images from the microfluidic circuit, wherein themicrofluidic circuit is located between the excitation filter and theemission filter.
 49. The portable diagnostic'system of claim 47, whereinthe analytes are detected at clinically relevant levels.
 50. Theportable diagnostic system of claim 47, wherein multiple analytes aredetected in parallel.